Bi-functional alkylating agents are important components of many chemotherapeutic regimens that form a variety of DNA damages including mono-adducts, intra- and interstrand DNA cross-links (ICL). Interstrand cross-links are among the most deleterious lesions reminiscent of DNA double strand breaks, because they constitute absolute blockades to DNA replication, recombination, and gene transcription. While mechanisms for repairing mono-adducts and intrastrand cross-links have been studied extensively, little is known about the components and the mechanisms of interstrand cross-linking repair in humans due to the lack of lesion-specific assays. We have developed reporter reactivation-based approaches to examine specifically the repair processes of DNA cross-links in mammalian cells. Our hypothesis is that both homology-dependent and -independent mechanisms may be involved in the processing of DNA interstrand cross-links. Our accomplished studies have provided evidence for a recombination-independent mechanism for ICL removal in human cells. In the present proposal, we will like to accomplish three goals. 1. Determine the effect of transcription and replication on homology-independent ICL repair; 2. Explore the role of human lesion-bypass polymerase Rev3 in ICL repair through generation of gene silencing models. 3. Establish an in vivo model for the homologous recombinational repair of ICLs. These studies should generate informative results on the repair mechanisms of DNA interstrand cross-links in mammalian cells and allows us to better understand the mutagenic consequences of DNA alkylating chemotherapeutic agents.